Cyclin D mediates tolerance of genome-doubling in cancers with functional p53.

Background: Aneuploidy and chromosomal instability (CIN) are common features of human malignancy that fuel genetic heterogeneity. Although tolerance to tetraploidization, an intermediate state that further exacerbates CIN, is frequently mediated by TP53 dysfunction, we find that some genome-doubled tumours retain wild-type TP53. We sought to understand how tetraploid cells with a functional p53/p21-axis tolerate genome-doubling events. Methods: We performed quantitative proteomics in a diploid/tetraploid pair within a system of multiple independently derived TP53 wild-type tetraploid clones arising spontaneously from a diploid progenitor. We characterized adapted and acute tetraploidization in a variety of flow cytometry and biochemical assays and tested our findings against human tumours through bioinformatics analysis of the TCGA dataset. Results: Cyclin D1 was found to be specifically overexpressed in early but not late passage tetraploid clones, and this overexpression was sufficient to promote tolerance to spontaneous and pharmacologically induced tetraploidy. We provide evidence that this role extends to D-type cyclins and their overexpression confers specific proliferative advantage to tetraploid cells. We demonstrate that tetraploid clones exhibit elevated levels of functional p53 and p21 but override the p53/p21 checkpoint by elevated expression of cyclin D1, via a stoichiometry-dependent and CDK activity-independent mechanism. Tetraploid cells do not exhibit increased sensitivity to abemaciclib, suggesting that cyclin D-overexpressing tumours might not be specifically amenable to treatment with CDK4/6 inhibitors. Conclusions: Our study suggests that D-type cyclin overexpression is an acute event, permissive for rapid adaptation to a genome-doubled state in TP53 wild-type tumours and that its overexpression is dispensable in later stages of tumour progression.

Characterization of 17 novel polymorphic microsatellite loci in the mammal chewing louse Geomydoecus ewingi (Insecta: Phthiraptera) for population genetic analyses.

We report 17 novel microsatellite loci in the parasitic chewing louse Geomydoecus ewingi, a common parasite of the pocket gopher, Geomys breviceps . Thirty-three G. ewingi individuals from 1 geographic locality and 3 pocket gopher hosts (populations) were genotyped at each locus. The number of alleles per locus ranged from 3 to 13. Observed heterozygosity ranged from 0.182 to 0.788. Four to 6 loci per louse population fell outside of Hardy-Weinberg expectations (HWE) and examination of population structure also revealed substantial homozygote excess as well as significant structure among louse populations. These findings are likely the consequence of biological characteristics of the lice (low dispersal abilities, population bottlenecks, etc.), which can result in inbreeding. Notably, when all louse individuals were analyzed together as 1 population, a Wahlund effect was detected, supporting that louse populations are restricted to 1 host individual. The microsatellite markers characterized in this study will be useful in future studies exploring the population dynamics in host-parasite systems, potentially yielding a better understanding of the processes underlying symbiotic associations.

Does mating behaviour affect connectivity in marine fishes? Comparative population genetics of two protogynous groupers (Family Serranidae).

Pelagic larval duration (PLD) has been hypothesized to be the primary predictor of connectivity in marine fishes; however, few studies have examined the effects that adult reproductive behaviour may have on realized dispersal. We assessed gene flow (connectivity) by documenting variation in microsatellites and mitochondrial DNA sequences in two protogynous species of groupers, the aggregate spawning red hind, Epinephelus guttatus, and the single-male, harem-spawning coney, Cephalopholis fulva, to ask whether reproductive strategy affects connectivity. Samples of both species were obtained from waters off three islands (Puerto Rico, St. Thomas and St. Croix) in the Caribbean Sea. Despite the notion that aggregate spawning of red hind may facilitate larval retention, stronger signals of population structure were detected in the harem-spawning coney. Heterogeneity and/or inferred barriers, based on microsatellites, involved St. Croix (red hind and coney) and the west coast of Puerto Rico (coney). Heterogeneity and/or inferred barriers, based on mitochondrial DNA, involved St. Croix (coney only). Genetic divergence in both species was stronger for microsatellites than for mitochondrial DNA, suggesting sex-biased dispersal in both species. Long-term migration rates, based on microsatellites, indicated asymmetric gene flow for both species in the same direction as mean surface currents in the region. Red hind had higher levels of variation in microsatellites and lower levels of variation in mitochondrial DNA. Long-term effective size and effective number of breeders were greater for red hind; estimates of θ(f) , a proxy for long-term effective female size, were the same in both species. Patterns of gene flow in both species appear to stem in part from shared aspects of larval and adult biology, local bathymetry and surface current patterns. Differences in connectivity and levels of genetic variation between the species, however, likely stem from differences in behaviour related to reproductive strategy.

Spawning frequency of brood dams and sires in a marine fish stock-enhancement hatchery.

Parentage analysis, employing five hypervariable microsatellite markers, was used to follow spawning patterns of red drum Sciaenops ocellatus broodfish in two spawning tanks through most of a calendar year in a marine fish hatchery dedicated to stock enhancement. Five of six dams and all four sires spawned at least once during the year. Variation in dam and sire spawning incidence and in number of progeny produced per dam and per sire translated into reduced genetic effective size (N e) per spawn by 40·6% in one tank and 50·8% in the other.

A genetic linkage map of red drum, Sciaenops ocellatus.

Second-generation, sex-specific genetic linkage maps were generated for the economically important estuarine-dependent marine fish Sciaenops ocellatus (red drum). The maps were based on F(1) progeny from each of two single-pair mating families. A total of 237 nuclear-encoded microsatellite markers were mapped to 25 linkage groups. The female map contained 226 markers, with a total length of 1270.9 centiMorgans (cM) and an average inter-marker interval of 6.53 cM; the male map contained 201 markers, with a total length of 1122.9 cM and an average inter-marker interval of 6.03 cM. The overall recombination rate was approximately equal in the two sexes (♀:♂=1.03:1). Recombination rates in a number of linkage intervals, however, differed significantly between the same sex in both families and between sexes within families. The former occurred in 2.4% of mapped intervals, while the latter occurred in 51.2% of mapped intervals. Sex-specific recombination rates varied within chromosomes, with regions of both female-biased and male-biased recombination. Original clones from which the microsatellite markers were generated were compared with genome sequence data for the spotted green puffer, Tetraodon nigroviridis; a total of 43 matches were located in 17 of 21 chromosomes of T. nigroviridis, while seven matches were in unknown portions of the T. nigroviridis genome. The map for red drum provides a new, useful tool for aquaculture, population genetics, and comparative genomics of this economically important marine species.

PCR amplification of microsatellites from single cells of Karenia brevis preserved in Lugol's iodine solution.

A simple and effective protocol is described for multiplex polymerase chain reaction (PCR) amplification of single cells of Karenia brevis. The protocol requires minimum processing, avoids additions that might dilute target DNA template, and can be used on cells preserved in Lugol's iodine preservative. Destaining of Lugol's-preserved cells with sodium thiosulfate allowed successful amplification of single-copy, nuclear-encoded microsatellites in single cells of K. brevis that have been preserved for up to 6 years.

Application of hypervariable genetic markers to forensic identification of 'wild' from hatchery-raised red drum, Sciaenops ocellatus.

Forensic identification of 'wild' versus hatchery-produced (cultured) red drum (Sciaenops ocellatus), an economically important marine fish in the southern United States, was assessed using hypervariable nuclear-encoded microsatellites and sequences of mitochondrial DNA. Both genotype exclusion and likelihood-ratio tests successfully identified 'wild' and 'cultured' individuals within requisite error bounds and within the context of complete parental sampling. Of the two, genotype exclusion was more effective, producing satisfactory results with fewer microsatellites and larger allowable error rates. Assignment tests proved ineffective, most likely because of the low level of genetic divergence between the sampled populations. An optimal, minimum set of ten markers that will reduce potential genotyping costs is identified. Results of the study should allay concerns regarding identification of 'wild'-caught fish sold illegally.

Specialty audit leads--has this concept been effective in implementing clinical audit in an acute hospital?

Clinical audit has a pivotal role to play in improving the quality of patient care. As part of the programme for co-ordinating clinical audit across the trust each clinical area has a nominated doctor to lead and co-ordinate the audit programme in their specialty. This qualitative survey reviews the effectiveness of having specialty audit leads and reviews the progress that specialties have made in developing their audit programme. The semi-structured interviews with 30 clinical audit leads identified an uneven level of development of clinical audit across the trust, and demonstrated that dedicated time would be needed to make these posts more universally successful. Although one size will not fit all, the interviews highlighted some recurring themes--seeds of success--in functioning audit programmes.

Heterosubtypic immunity against human influenza A viruses, including recently emerged avian H5 and H9 viruses, induced by FLU-ISCOM vaccine in mice requires both cytotoxic T-lymphocyte and macrophage function.

Induction of heterosubtypic immunity to influenza viral antigens is of paramount importance to the prevention of epidemics and potential pandemics. The 1997 incidence of avian influenza infections in humans in Hong Kong heightened the need for pandemic preparedness and a search for vaccines and vaccine delivery systems that can confer broad protection. In this report, we demonstrate that the delivery of H1N1 subtype influenza viral antigens as immunostimulating complexes (ISCOM) induces broad cross-protection in mice against challenge with various influenza virus subtypes, including the avian H9 and the H5 strains that were recently responsible for deaths in humans. The ISCOM delivery system induced high and long-lived serum antiviral antibodies and class I-restricted cytotoxic T-lymphocytes (CTL). Studies with perforin, IFN-gamma, and mu-chain gene knock-out mice demonstrated that the heterosubtypic protection required cross-reactive, functional cytotoxic T cells and nonhemagglutination inhibiting serum antibodies. Interferon-gamma, a major player in viral clearance by nonlytic mechanisms, did not appear to play a role in heterosubtypic immunity. Nonformulated H1N1 influenza antigens failed to induce significant CTL or long-lasting antibody responses or to protect mice against challenge with heterosubtypic viruses. Furthermore, while influenza virus infection induced a dominant nucleoprotein (NP)-specific CTL response in H2 mice, the ISCOM delivery system induced a dominant hemagglutinin-specific CTL response. Moreover, non-neutralizing but cross-reactive antibodies played a role in reducing viral titers by macrophages. These results suggest that exogenous delivery of influenza antigens as ISCOM can influence their antigen processing and presentation, their ability to induce/recall CTL specificities, and their capacity to mediate broad cross-protection against influenza virus variants.

Activated protein C induction of MCP-1 in human endothelial cells: a possible role for endothelial cell nitric oxide synthase.

Classically, activated protein C (APC) of the protein C/protein S anticoagulant pathway has functioned not only to inactivate the procoagulant factors Va and VIIIa but also to inhibit the activity of plasminogen activator inhibitor-1 (PAI-1). More recent data have suggested that the protein C/protein S pathway may serve as a physiological link between coagulation and inflammation. This APC pathway link was proposed because of observations showing that APC could both modulate the effects of cytokines and block neutrophil activation. As a further extension of the effect(s) of APC on cytokines, we found that APC, at the equivalent physiological protein C concentration of 4 microg/ml, significantly upregulated monocyte chemotactic protein-1 (MCP-1) RNA in human umbilical vein endothelial cells (HUVECs), as indicated by a ribonuclease protection assay (RPA) at 3 and 6 h with a return to near basal levels by 24 h. ELISA determinations demonstrated that 4 microg/ml of APC induced a significant (P=.0001) increase in MCP-1 protein production over basal levels within a 24-h period. At the same concentration, APC downregulated endothelial cell nitric oxide synthase (eNOS) RNA. Downregulation first became apparent at 6 h and continued through 48 h of culture. This downregulation was concentration dependent over a range of 1.3-12 microg/ml, and there was no effect on cell viability within this range. In support of other studies, we also found that exogenously added nitric oxide (NO) inhibited MCP-1 production. These data suggest that APC may induce MCP-1 through the inhibition of eNOS.

Fluorescent multiplex polymerase chain reaction analysis of four genes associated with inpaired fibrinolysis and myocardial infarction.

Multiplex polymerase chain reaction (PCR) allows for the simultaneous amplification of several genes, thereby optimizing the use of reagents and decreasing personnel time. Multiplex PCR was used to amplify four genes in one PCR reaction, demonstrating the advantage of multiplex PCR for our study since it allowed us to amplify four separate genes using only 1 microl DNA, thus maximizing the use of study DNA. As compared with conventional multiplex PCR analysis with ethidium bromide, incorporating fluorescence-labeled primers into multiplex PCR reactions facilitated accurate, simultaneous analysis of many DNA fragments within one base discrimination. We have used this fluorescence methodology to analyze polymorphisms associated with either impaired fibrinolysis or myocardial infarction. These include the angiotensin converting enzyme insertion/deletion (I/D) polymorphism in intron 16 of the DCP1 gene, the Alu I/D polymorphism of the tissue plasminogen activator-25 locus in intron 8, the plasminogen activator inhibitor 4G/5G repeat polymorphism, and the variable number tandem repeat of the endothelial cell nitric oxide synthase gene, all characterized by an insertion, deletion, or repeat. The amplified products were diluted 1 : 60 and analyzed on the ABI PRISM 310 Genetic Analyzer using GeneScan software. With this method, we were able to amplify four genes using 75% less reagents and personnel time, thus demonstrating the benefit of multiplex PCR and fluorescence technology.

Mutations in the genes regulating methylene tetrahydrofolate reductase (MTHFR C-->T677) and cystathione beta-synthase (CBS G-->A919, CBS T-->c833) are not associated with myocardial infarction in African Americans.

Moderate hyperhomocysteinemia is a putative risk factor for cardiovascular disease. Molecular studies have demonstrated increased plasma homocysteine levels in the presence of DNA mutations in either the methylenetetrahydrofolate reductase (MTHFR) enzyme found in the remethylation pathway or the enzyme cystathione beta-synthase (CBS) of the transsulfuration pathway. To determine whether the mutation C-->T677 in the MTHFR gene or the T-->C833/844ins68 and G-->A919 mutations in the CBS gene are associated with myocardial infarction (MI) in African Americans, DNA was analyzed from samples obtained from a case-control study conducted at a large, inner-city hospital. One-hundred ten African American subjects with a diagnosis of MI and 185 race- and age-matched controls were recruited. Our results demonstrated that 15% of the MI cases were heterozygous for the C-->T677 (MTHFR) mutation, while 1.8% were homozygous. When compared to the controls in which 15% were heterozygous and 2.1% were homozygous, no significant association with MI was observed. In addition, 34% of the cases were heterozygous for the T-->C833 (CBS) mutation while 6% were homozygous. This is compared to 32% and 5% of the controls having the heterozygous and homozygous genotype, respectively. No significant association was observed for the T-->C833 (CBS) mutation among the cases and controls. Although this mutation has no significant association with MI, the prevalence of the heterozygous state was higher than what has been reported for whites (12%). No mutations for G-->A919 (CBS) were detected in the cases or controls. The racial differences of the CBS T-->C833 polymorphism suggest that further investigation into the other areas of the CBS gene is needed.

Expression of chemokine by human coronary-artery and umbilical-vein endothelial cells and its regulation by inflammatory cytokines.

BACKGROUND: Activation of the endothelium is a critical event in the process of inflammation and is associated with the production of chemokines. OBJECTIVE: To evaluate the proinflammatory cytokine-induced chemokine repertoire of human coronary-artery endothelial cells (HCAEC) both at the messenger RNA (mRNA) level and at protein level in direct comparison with that of human umbilical-vein endothelial cells (HUVEC). METHODS: Human coronary-artery and human umbilical-vein endothelial cells were obtained commercially and experimental data were derived from cell cultures between passage levels 3 through 6. Supernatant fluids from cytokine [tumor necrosis factor-alpha (TNF-alpha), interleukin-1-alpha, and anti-TNF R55] stimulated endothelial cell cultures were used to study chemokine release. Sandwiched ELISA assays, obtained commercially, were used to estimate cell culture supernatant fluid levels of the selected chemokines: monocytic chemotactic protein-1, regulated upon activated normal T cells expressed and secreted, interleukin-8, transforming growth factor-beta-2 (TGF-beta2), and gamma interferon protein-10. Expression of messenger RNA was determined using selected labeled riboprobes (32P UTP) in a ribonuclease protection assay using total cellular mRNA. RESULTS: Upon in-vitro stimulation with TNF-alpha and interleukin-1-alpha, production of regulated-upon-activated-normal-T-cells expressed and secreted (RANTES) protein by HCAEC was significantly increased relative to that by HUVEC, the greatest effect being found with interleukin-1-alpha. The opposite effect, however, was noted for levels of monocytic-chemotactic-protein-1 protein, which were detected in HUVEC at significantly higher levels than they were in HCAEC challenged by those cytokines. Production of gamma interferon-inducible protein-10 (gammaIP-10) by HUVEC was induced by TNF-alpha and interleukin-1-alpha, whereas only a modest induction by interleukin-1-alpha was seen in HCAEC. TGF-beta-2 protein was constitutively expressed in HCAEC but not in HUVEC. Expression of mRNA was analyzed by the ribonuclease-protectionassay. RANTES mRNA was expressed in HCAEC from 3 h through 48 h after treatment with TNF-alpha, whereas only a modest induction of RANTES was expressed in HUVEC 24 h and 48 h after treatment with TNF-alpha. Monocytic-chemotactic-protein-1 mRNA was constitutively expressed by both types of cell, but the basal levels in HCAEC was significantly higher than in HUVEC. HCAEC constitutively expressed both TGF-beta-1 and TGF-beta-2 mRNA, whereas HUVEC constitutively expressed TGF-beta-1 only. CONCLUSION: Our data indicate that HCAEC and HUVEC express chemokines differently, which could contribute to or influence site-specific recruitment of subsets of leukocytes.

Mucosal delivery of inactivated influenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal influenza A H5N1 virus infection.

Influenza vaccines that induce greater cross-reactive or heterosubtypic immunity (Het-I) may overcome limitations in vaccine efficacy imposed by the antigenic variability of influenza A viruses. We have compared mucosal versus traditional parenteral administration of inactivated influenza vaccine for the ability to induce Het-I in BALB/c mice and evaluated a modified Escherichia coli heat-labile enterotoxin adjuvant, LT(R192G), for augmentation of Het-I. Mice that received three intranasal (i.n.) immunizations of H3N2 vaccine in the presence of LT(R192G) were completely protected against lethal challenge with a highly pathogenic human H5N1 virus and had nasal and lung viral titers that were at least 2,500-fold lower than those of control mice receiving LT(R192G) alone. In contrast, mice that received three vaccinations of H3N2 vaccine subcutaneously in the presence or absence of LT(R192G) or incomplete Freund's adjuvant were not protected against lethal challenge and had no significant reductions in tissue virus titers observed on day 5 post-H5N1 virus challenge. Mice that were i.n. administered H3N2 vaccine alone, without LT(R192G), displayed partial protection against heterosubtypic challenge. The immune mediators of Het-I were investigated. The functional role of B and CD8+ T cells in Het-I were evaluated by using gene-targeted B-cell (IgH-6(-/-))- or beta2-microglobulin (beta2m(-/-))-deficient mice, respectively. beta2m(-/-) but not IgH-6(-/-) vaccinated mice were protected by Het-I and survived a lethal infection with H5N1, suggesting that B cells, but not CD8+ T cells, were vital for protection of mice against heterosubtypic challenge. Nevertheless, CD8+ T cells contributed to viral clearance in the lungs and brain tissues of heterotypically immune mice. Mucosal but not parenteral vaccination induced subtype cross-reactive lung immunoglobulin G (IgG), IgA, and serum IgG anti-hemagglutinin antibodies, suggesting the presence of a common cross-reactive epitope in the hemagglutinins of H3 and H5. These results suggest a strategy of mucosal vaccination that stimulates cross-protection against multiple influenza virus subtypes, including viruses with pandemic potential.

Immunity to influenza A H9N2 viruses induced by infection and vaccination.

Avian influenza A H9N2 viruses are widespread among domestic poultry and were recently isolated from humans with respiratory illness in China. Two antigenically and genetically distinct groups of H9N2 viruses (G1 and G9) are prevalent in China. To evaluate a strategy for vaccination, we compared G1 and G9 viruses for their relative immunogenicity and cross-protective efficacy. Infection of BALB/c mice with representative viruses of either group protected against subsequent challenge with the homologous or heterologous H9N2 virus in the absence of detectable cross-reactive serum hemagglutination inhibition antibody. Mice injected intramuscularly with inactivated G1 whole virus vaccine were completely protected from challenge with either H9N2 virus. In contrast, mice administered inactivated G9 vaccine were only partially protected against heterologous challenge with the G1 virus. These results have implications for the development of human vaccines against H9N2 viruses, a priority for pandemic preparedness.

Death effector domain protein PEA-15 potentiates Ras activation of extracellular signal receptor-activated kinase by an adhesion-independent mechanism.

PEA-15 is a small, death effector-domain (DED)-containing protein that was recently demonstrated to inhibit tumor necrosis factor-alpha-induced apoptosis and to reverse the inhibition of integrin activation due to H-Ras. This led us to investigate the involvement of PEA-15 in Ras signaling. Surprisingly, PEA-15 activates the extracellular signal receptor-activated kinase (ERK) mitogen-activated protein kinase pathway in a Ras-dependent manner. PEA-15 expression in Chinese hamster ovary cells resulted in an increased mitogen-activated protein kinase kinase and ERK activity. Furthermore, PEA-15 expression leads to an increase in Ras guanosine 5'-triphosphate loading. PEA-15 bypasses the anchorage dependence of ERK activation. Finally, the effects of PEA-15 on integrin signaling are separate from those on ERK activation. Heretofore, all known DEDs functioned in the regulation of apoptosis. In contrast, the DED of PEA-15 is essential for its capacity to activate ERK. The ability of PEA-15 to simultaneously inhibit apoptosis and potentiate Ras-to-Erk signaling may be of importance for oncogenic processes.

The role of the t-PA I/D and PAI-1 4G/5G polymorphisms in African-American adults with a diagnosis of myocardial infarction or venous thromboembolism.

To determine whether or not the PAI-1 4G/5G and t-PA I/D polymorphisms in African-Americans were linked to cardiovascular disease, the association of these polymorphisms to disease expression was analyzed in a recently completed case-control study of myocardial infarction or venous thromboembolism among African-Americans. All African-Americans patients with a history of venous thromboembolism attending an anticoagulant clinic, and patients with a history of a MI attending a cardiology clinic at a large local urban public hospital were eligible for inclusion as cases in the study. In this study it was observed that there was a statistically significant association between the D allele of the t-PA I/D polymorphism and venous thromboembolism and a nonsignificant association between the D allele and myocardial infarction among African-Americans. t-PA antigen levels were statistically significantly higher among both myocardial infarction and venous thromboembolism cases compared with control subjects. The genotypes were unrelated to t-PA plasma levels. There was no association between either myocardial infarction or venous thromboembolism and the 4G/5G PAI-1 genotype. It was also found that genotype frequencies for both PAI-1 4G/5G and t-PA I/D polymorphisms in African-American adults were different from those reported for both U.S. Causcians and Europeans.

The c-Abl tyrosine kinase contributes to the transient activation of MAP kinase in cells plated on fibronectin.

Previous work showed that integrin stimulation triggers activation of the c-Abl tyrosine kinase and its transient localization to focal adhesions. We now report that plating cells on fibronectin triggers association of Grb2 with c-Abl, suggesting possible involvement of c-Abl with integrin activation of the MAP kinase pathway. Expression of a kinase-defective c-Abl specifically inhibited the transient induction of Erk2 activity following cell adhesion. Together with the known ability of activated, oncogenic forms of c-Abl to activate Ras and the MAP kinase pathway, these data suggest that c-Abl contributes to the integrin induction of MAP kinase activity.

Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase.

The mitogen-activated protein (MAP) kinase pathway is a critical regulator of cell growth, migration, and differentiation. Growth factor activation of MAP kinase in NIH 3T3 cells is strongly dependent upon integrin-mediated adhesion, an effect that contributes to the anchorage dependence of normal cell growth. We now show that expression of constructs that constitutively activate focal adhesion kinase (FAK) rescued the defect in serum activation of MAP kinase in suspended cells without directly activating MAP kinase. Dominant negative FAK blocked both the rescue of suspended cells by the activated construct and the serum activation of MAP kinase in adherent cells. MAP kinase in FAK(-/)- mouse embryo fibroblasts was adhesion-insensitive, and reexpression of FAK restored its adhesion dependence. MAP kinase activity in ras-transformed cells is still decreased in suspension, but expression of constructs that constitutively activate FAK enhanced their anchorage-independent growth without increasing adherent growth. V-src, which activates both Ras and FAK, induced MAP kinase activation that was insensitive to loss of adhesion, and that was blocked by a dominant negative FAK. These results demonstrate that FAK mediates the integrin requirement for serum activation of MAP kinase in normal cells, and that bypassing this mechanism contributes to anchorage-independent growth in transformed cells.